The Experimental And Clinical Study Of Dot-blot Hybridization In Diagnosis Of Invasive Aspergillosis

Study Background and PurposeWith the development of medicine in recent years,the treatment methods of various diseases are constantly innovating,such as hematopoietic stem cell transplantation,organ transplantation,cancer chemotherapy and the extensively development of the indwelling catheter technique;Widely use of broad-spectrum antibiotics,corticosteroids and immunosuppressive,as well as the growing AIDS population.As a result of these reasons,the rate of infecting by the conditions pathogenic fungi is rising and the spectrum of fungal pathogens has also changed. At present,the common clinical practice of deep fungus is Candida,Aspergillus, Cryptococcus and so on.Candida accounts for about 40%～70%Aspergillus infection in recent years have significantly higher than in previous years, particularly in the absence of neutrophils and bone marrow transplant patients,The infection rate is up to 50%and it is the largest and the worst prognosis of deep fungal infection,and high mortality,the most common and most pathogenic bacteria is aspergillus fumigatusExploring the early,specific and sensitive rapid diagnostic method of fungal infection has been a hot and difficult topic in research area.At present,people focus on two areas of Aspergillus antigen in serum and molecular biology for rapid detection.Existing serological methods,antibody detection methods are limited due to immunosuppression patients cannot reflect the level of antibodies.Although antigen detection methods have advantages,the inadequacy of methods of study having not been extensively carried out.The recent emergenced method of galactomannan(GM) detection with ELISA,has good sensitivity and been extensively carried out in Europe and the United States,but it is too expensive to be widely used in our country.In addition,patients who are lack of neutrophils in the blood have antigen-presenting cell and lymphocytes functional lesion,making serological tests(such as galactomannan test,etc.),loss of their diagnostic value.The development of molecular biology has opened up broad prospects for the detection of pathogenic microorganisms,PCR amplification and molecular hybridization detection methods are the basic technologies,combining these two technologies we have designed a species-specific primers for Aspergillus fumigatus PCR amplification,The PCR product as a probe tagged with digxin and dot-blot hybridization with the amples of fungal DNA.With it can we set up an early,rapid, sensitive,specific and widely carried out for clinical Aspergillus fumigatus DNA detection methods..We conducted the following studies:1.Seting up method of hybridization by species-specific long-chain nucleotide probe:design specificity primers for Aspergillus fumigatus alkaline protease and polymerase chain reaction of DNA for about 465bp,labeled with digoxigenin.Aspergillus fumigatus, Aspergillus flavus,Candida,Cryptococcus and other fungal and bacterial DNA fixed on nylon membrane dot blot hybridizated with digoxigenin-labeled probe, positive hybridization results can be make sure fumigatus fungal infection.2. Conducting invasive Aspergillus fumigatus rat model:collecting serum and Bronchoalveolar lavage fluid(BALF) specimens and extracting DNA.Evaluation the diagnostic value of long-chain probe dot-blot hybridization method in serum and bronchoalveolar lavage fluid in animal models.3.Detected by dot-blot hybridization of clinical specimens in patients with invasive aspergillosis to evaluate the dot blot technique in invasive aspergillus infection in clinical diagnostic value.Methods1.Aspergillus species-specific probes Preparation:Fungal DNA extraction:DNA extraction using CTAB method.Aspergillus fumigatus species-specific probes(ALP) gene primers:Up primer 5'-TCCGTGTACTTGATGGGTCT-3Down primer 5'-ATGTACGAGGATGTGAGCCG-3primer with reference to the literature reported the nucleotide sequence.ALP gene PCR amplification product analysis and sequencing:ALP gene was amplified by PCR,the fragment length is 465bp.Product by agarose gel electrophoresis and sequencing Sequencing results compared with ALP gene nucleotide sequences provided by GenBank and determine their genetic.2.Probe marking and dot-blot hybridization:ALP gene PCR amplification products tagged by digoxigenin using random primer method.Aspergillus fumigatus,Aspergillus flavus,Candida,Cryptococcus and other fungal and bacterial DNA fixed on nylon membrane dot blot hybridization with digoxigenin-labeled probe,positive results can be make sure infected with aspergillus fumigatus..3.Establish animal model of IA infection72 SD rats were divided into three groups,the experimental group 48 examples, the normal control group and immunosuppressive control group 12 examples respectively.Establish immunesuppression and infection animal model: The experimental group and immunosuppression control group,rats were given intramuscular dexamethas 0.6mg/kg,cyclophosphamide 75mg/kg,consecutive 2 days.On the third days,the experimental group were given intramuscular injection of dexamethasone 0.6mg/kg,then 1×10~8/ml Aspergillus fumigatus spore suspension slow trickle-down dual-nostril of rats,and maintained rats upright.Trickle-down suspension for about 0.3～0.4 ml,one times a day,for three days. Immunosuppression control group given intramuscular dexamethasone 0.6mg/kg, by dual-nostril instillation of normal saline about 0.3～0.4 ml,one times a day,for three days.Serum,lavage fluid and viscera samples collection and treatment:Animals were killed on the first,third,fifth,seventh day,at the same time collect the serum and BALF preserved at-20℃.Organs as lung,liver,lien were get out and make part of these tissue as homogenate,inoculated into Sabouraud plate cultivate more than 96h.Put the remaining oragns tissue in 10%formalin fixed for more than eight hours, paraffin-embedded,and dyed withHE andPASM to detect the shape of aspergillus fumigatus.4.Dot-blot hybridization test specimens of animal model:Fungal DNA of serum,BALF samples extraction methods are referenced to the literature.Fix the samples DNA on the nylon and dot-blot hybridization with the long-chain Aspergillus fumigatus probe.Compare experimental group with control group,while compare dot-blot hybridization with culture.5.The collection of clinical specimens and testing12 cases with high-risk invasive aspergillus were collected 8 cases of male, female 4 cases.Among them pathological diagnosis aspergillus infection was 2 case, clinical diagnosis aspergillus infection was 2 cases,eight cases were suspected. Collected serum samples of these patients,low-temperature opreservation then tested using dot blot hybridization.which set up in the first two parts of this article.6.Statistical Analysis Methods:SPSS 13.0 statistical software,PearsonX~2 test,mult-sample comparison of the rateThe results1.Aspergillus fumigatus alkaline protease(ALP)gene PCR amplification results:PCR amplification the Aspergillus fumigatus,Aspergillus flavus,Aspergillus terreus,Aspergillus niger,Candida albicans,Pseudomonas aeruginosa,human whole blood cell DNA by Aspergillus fumigatus-specific primers,and electrophoresis.The results show that only Aspergillus fumigatus expanding a specific band of 465bp,the other fungi,bacteria were no specific amplification band. Indicates that the pairs of primers for Aspergillus fumigatus are highly specific. Conducts sequencing result is 99%similarity with gene library NC.007197.11.2.Dot blot Hybridization results:Specificity:The DNA extracted from Aspergillus fumigatus,Aspergillus flavus, Aspergillus niger,Aspergillus terreus,Candida albicans,Staphylococcus aureus, hepatitis B virus,human whole blood cell were preparated to 10mg/ml,take out 3μl and fixed on nylon membrane,respectively,and Dot blot Hybridization with the digoxigenin labeled Aspergillus fumigatus-specific hybridization probe.The results showed that this probe has a high specificity,that is,only with Aspergillus fumigatus DNA hybridization,and other fungal,bacterial DNA without cross-reaction.Sensitivity:The DNA extracted from Aspergillus fumigatus-fold diluted, respectively,than point on the nylon membrane,and Dot blot hybridization with Aspergillus fumigatus probe It can detected in the amount of DNA as little as100fg.3.Conduction Invasive infection model:Organ tissue culture and blood culture:the experimental group of animals autopsy:the liver swelling,the surface can be see a lot of abscess point,the lung tissue surface can be seen much small nodule,others visceral no abscess points and nodules.Organ tissue culture can be seen the green colony,the texture was villous, the back was light yellow.Normal control group and the immunosuppression control group:organ culture had no growth of Aspergillus fumigatus.Blood culture were no fungal growth.Oragan tissues HE dyeing and PAS dyeing:the experimental group histopathological sections stained by HE,lung and liver tissue shows inflammatory cell infiltration,tissue necrosis,biopsy for methenamine silver (PASM) staining can be seen in the necrotic tissue in piles of mycelium,which was strip has separated branch for the acute angle.Cultivate fungi histological biopsy and organizations methenamine silver staining confirmed Aspergillus fumigatus, invasive aspergillosis successfully produced an animal model.Normal control group and the control group immunosuppression:HE dyeing tissues and PASM dyeing were normal.4.Specimens of animal model test resultsExtracting DNA from the collected serum and lavage specimens and dot-blot hybridization,the positive rate of the experimental group is higher than the normal control group and the immune control group(P＜0.05),statistically significant difference.With the time of infection,the positive rate of infection is increased.On the first days after infection the experimental group and the immune suppression and normal control group were not significantly different(P＞0.00625),The third days after infection,serum samples of the immunosuppression control group were also no significant difference from experimental group(P＞0.00625).The other groups were statistically significant differences(P＜0.00625).The positive rate of blood culture and culture-positive rate of BALF were lower than hybridization,but required for long time5.Clinical specimens test results12 cases with high-risk invasive aspergillus were collected 8 cases male,4 cases of female.Among them pathological diagnosis aspergillus infection was 2 case,clinical diagnosis aspergillus infection was 4 cases,6 cases were suspected. Collected serum samples of these patients before treatment and extracts DNA,then tested using dot blot hybridization.which set up in the first two parts of this article,5 cases of positive test results.Diagnosis and clinical diagnosis of Aspergillus infection of dot hybridization detection of serum were positive.Conclusion1.Successfully set up a dot-blot hybridization technology for detection Aspergillus infection:design the Aspergillus fumigatus-specific probes have a high degree of specificity,with other fungi,bacteria,no cross-reaction,this method can be identified the species of Aspergillus.With high sensitivity,it can detect as little as 100fg fungal DNA.2.Completed the performance evaluation of dot-blot hybridization in animal models and invasive high-risk patients clinical specimens:succefully established Aspergillus fumigatus infection animal models by trickle-down the nostrilss spores. Organ damage lungs,liver obviously.Dot-blot hybridization can be detected serum and bronchoalveolar lavage fluid of animal models in the small amount of fungal DNA,sensitivity is 67.6%,specificity is 89.6%.The positive rate of the experimental group was significantly higher than control group. 3.Evaluation the value of Dot-blot hybridization on clinical:Clinical specimen of high-risk patients,confirmed the diagnosis of patients and clinical specimens were positive by dot blot hybridization detection.